Almost fifty years ago, oral administration of Bis-dichloroacetyl-diamines (BDADs) such as WIN 18,446 was shown to safely, completely and reversibly inhibit spermatogenesis in men. These compounds were not brought to market as male contraceptives, however, because they caused a "disulfiram reaction" characterized by flushing, nausea and vomiting when co-ingested with alcohol. Further research in this area was stalled by lack of knowledge regarding the mechanism by which BDADs caused these effects. The disulfiram reaction is now known to be caused by the inhibition of the liver enzyme aldehyde dehydrogenase (ALDH2), normally involved in the metabolism of alcohol. The triggering of a disulfiram reaction when BDADs and alcohol are mixed suggests that BDADs also inhibit ALDH2. It seems plausible that BDADs also mediate their effects on spermatogenesis via inhibition of an aldehyde dehydrogenase. Within the seminiferous tubules of the testes, a testes-specific aldehyde dehydrogenase called ALDH1a2 biosynthesizes retinoic acid, a vitamin-A derivative known to be essential for spermatogenesis. Therefore, we hypothesize that BDADs such as WIN 18,446 suppress spermatogenesis by inhibiting the biosynthesis of retinoic acid by ALDH1a2 within the seminiferous tubules of the testes. In specific aim #1 of this proposal, we will endeavor to demonstrate that the mechanism by which BDADs such as WIN 18,446 suppress spermatogenesis involves inhibition of the formation of intratesticular retinoic acid. This will be accomplished in vitro using primary cell cultures of neonatal spermatogonia, as well as in vivo using the vitamin-A deficient mouse model and normal rabbits. In specific aim #2, we will endeavor to demonstrate that WIN 18,446 specifically inhibits ALDH1a2 using cell lines stably transduced with a cDNA encoding this enzyme. Next, in specific aim #3, we will synthesize novel derivatives of WIN 18,446 that specifically inhibit ALDH1a2 while minimizing inhibition of ALDH2-mediated alcohol metabolism. We will then examine the ability of these novel compounds to inhibit spermatogenesis both in vitro and in vivo. This work will provide insight into the physiology of spermatogenesis and result in substantial progress towards the development of a safe and effective oral, non-hormonal, reversible contraceptive for men. PUBLIC RELEVANCE: Despite currently available contraceptives, the world's population exceeds six and a half billion and is increasing by 80 million yearly. Much of this population growth is unintended and is due to inadequate contraception. Currently, male-directed contraceptive options are particularly limited. The research described in this proposal may eventually allow for the development of a safe and effective oral approach to male contraception, which will serve to greatly decrease the risk of unintended pregnancy and population growth.